CN1132928C - Two-saccharomycetes strains and its usage in preparing optically pure 2-aryl propionic acid - Google Patents

Abstract

The present invention discloses two yeast strains, Saccharomyces solidis CGMCC No.0573 and Trichospora brassicae CGMCC No.0574, and the use of their cells or esterases isolated therefrom to racemize 2-arylpropionic acid The esters undergo one or two cascaded hydrolysis reactions to produce (S) and (R)-2-arylpropionic acids with high optical purity. The present invention can not only simultaneously obtain (S) and (R)-type 2-arylpropionic acid single enantiomer products with very high optical purity, satisfying the needs of different purposes of medicine or pesticide industry, but also as the reaction substrate 2 -Ethyl aryl propionate is easy to synthesize without using expensive or toxic reagents, thereby reducing the cost of raw materials; in addition, it also saves the racemization and recycling of the remaining esters of the reaction, thereby simplifying the operation steps and improving the production efficiency. Raw material utilization.

Description

Two kinds of yeast strains and their method for preparing optically pure 2-arylpropionic acid

technical field

The present invention relates to yeast strains and a method for preparing optically pure 2-arylpropionic acid using microorganisms.

technical background

2-Arylpropionic acid (expressed as Ar-C ^* H(CH ₃ )-COOH by the general formula, where Ar represents a substituent containing an aromatic ring, and C ^* represents a chiral carbon atom), is a very important class of chiral Active compounds have a very wide range of uses in the fields of medicine and pesticides. It has been found that the two enantiomers of 2-arylpropionic acids differ significantly in their biological activity. Taking ketoprofen [2-(3-benzoylphenyl)-propionic acid] as an example, its (S)-enantiomer is a highly efficient non-steroidal anti-inflammatory agent, and its anti-inflammatory activity ratio ( R)-enantiomer is more than 100 times higher, while (R)-ketoprofen has the effects of analgesia and prevention of osteoporosis. Similar chirality also exists in other 2-arylpropionic acid NSAIDs (such as naproxen, ibuprofen, fenbufen, etc.) and 2-aryloxypropionic acid series herbicides effect.

Various chiral 2-arylpropionic acids synthesized by traditional organic chemistry methods are generally racemic mixtures of two enantiomers (R) and (S) in equal amounts. The classic method for the resolution of chiral carboxylic acids is to first form diastereomeric salts with a single isomer of chiral amines, and then use the difference in solubility to preferentially crystallize one isomer from solution. Since the diastereomeric crystallization method requires the use of expensive optically pure chiral resolving agents, the cost is relatively high, the process is complicated, and a large amount of waste water is generated. Selective transformation splitting methods are increasingly being developed and applied. Many documents and patents have been involved in this technology, and the typical ones are as follows:

(1) Use a commercial lipase preparation (mainly derived from Candida sp.) as a catalyst. Due to the easy source of commercial enzymes, there are many literature reports on this aspect, but when used to resolve 2-aryl propionic acid, especially ketoprofen, the stereoselectivity is often not ideal, so the enzyme must be purified or other additional processing. For example: document J.Am.Chem.Soc.1990, 112:1990-1995 is purified by enzyme and improves the selectivity of enzyme with deoxycholate and acetone treatment; Patent U.S.Pat.5 108 916, Apr.28, 1992 passes Purify the crude enzyme by ion exchange or isoelectric focusing to obtain a single component (CSC-1 or CSC-2) with high selectivity; the literature J.Org.Chem. directly treats the crude enzyme with 2-propanol to improve the three-dimensional Selectivity; the literature J.Am.Chem.Soc.1995, 117:6854 reported the effect of cross-linking enzyme crystals splitting chiral carboxylates. A common disadvantage of the above methods is that the price of commercial enzyme preparations is relatively high, and the additional treatment process is cumbersome and causes the loss of enzyme activity, thereby greatly increasing the cost of enzyme catalysts and affecting the practical feasibility of industrial applications.

(2) Stereoselective biotransformation of racemic substrates by direct use of microbial cells containing esterase. Because microbial cells are easy to cultivate, easy to operate, and low in cost, they are economically more advantageous than commercial enzymes. The key is to screen efficient and specific enzyme-producing strains. Typical documents in this respect have: U.S. Patent U.S.Pat.5 516 690, May14, 1996 discloses a strain of Trichosporon labbacchii, which uses ethanol as the only carbon source and separates it from soil; transforms it with its nutrient solution Ketoprofen ethyl ester can obtain the (S)-ketoprofen of 93% purity, if then can obtain (R)-ketoprofen with ketoprofen methyl ester as substrate, but enantioprofen The body purity is only 86%. If a product with higher purity is to be obtained (generally the purity of a pharmaceutical should reach more than 98%), it is also necessary to remove a small amount of residual enantiomeric impurities by diastereomeric crystallization. Another U.S. Patent U.S.Pat.5 457 051, Oct.10, 1995 disclosed a Beauveria bassiana ATCC44860 specially used for the preparation of (R)-ketoprofen, using its mycelium to convert keto For ibuprofen choline ester, the enantiomeric excess value of the obtained (R)-acid is only 93.6% ee (equivalent to 96.8% enantiomeric purity). Because the synthetic steps of ketoprofen choline ester are very complicated, and need to use poisonous reagent (as dimethyl sulfate and methyl iodide), therefore bring great difficulty to industrialized application.

In summary, when splitting 2-arylpropionic acid by biocatalysis, the price of commercialized enzyme preparations is high, the selectivity is not ideal, and additional processing steps need to be added, and the product of the reaction is usually (S )-acids and (R)-esters, the latter requires further chemical hydrolysis to convert to (R)-acids; in comparison, directly using enzyme-containing microbial cells to catalyze the separation of esters of chiral carboxylic acids, the process comparison Simple, relatively low cost, so it has good industrial application prospects. However, the currently disclosed microbial transformation technologies also have defects such as insufficient selectivity of enzyme-producing strains and inability to simultaneously obtain high-purity (S) and (R) two enantiomers. Contents of the invention

The technical problem to be solved in the present invention is to disclose two yeast strains with high stereospecificity and complementary enantioselectivity, and the use of their cells or the esterase isolated therefrom to racemize 2-arylpropane Ester carries out one or two series hydrolysis reactions, to prepare the method for (S) and (R)-2-aryl propionic acid of high optical purity, to overcome the selectivity that prior art exists is not high enough, and can't simultaneously Defects such as the two enantiomers (S) and (R) are obtained in high purity.

Design of the present invention is such:

(1) The types of microorganisms in nature are very rich and diverse. Since there are chiral esters with (R) and (S) configurations in nature, it can be inferred from the viewpoint of evolution that there must also be chiral esters with higher specificity for (R) and (S) substrates in nature. A single esterase. Therefore, the inventors believe that as long as a certain method is designed to skillfully screen and isolate highly specific esterase-producing bacteria, then through highly enantioselective ester hydrolysis, a highly optically pure single enantiomer 2- Aryl propionic acid.

(2) The enantiomer resolution process catalyzed by microorganisms and their enzymes is essentially a so-called "kinetic resolution" process based on the premise that the speed of the competitive reaction of two enantiomer substrates is different. The analysis and simulation curves have been described in detail in the document J.Am.Chem.Soc.1982, 104:7294. According to this theoretical model, the following enlightenment can be obtained: when the target enantiomer is the product (acid) of the reaction, firstly, the cell or enzyme with the highest possible enantioselectivity (E value) should be selected; secondly, in the cell or enzyme In the case that the selectivity is not particularly high (such as E<50), the time of the reaction should be controlled so that the conversion rate (c) of the reaction is appropriately low (for example, about 40%); After the body is enriched to a certain extent, another enzyme with opposite enantioselectivity is used for a second conversion, and the optical purity of the product obtained in the reaction is higher than that obtained when the racemic substrate is converted in one step with the latter enzyme alone. Optical purity should be high.

Microorganisms used:

For the preparation of optically pure 2-aryl propionic acid in the present invention is a kind of yeast belonging to the genus Citeromyces—Citeromyces matriensis CGMCC No.0573 and a yeast belonging to the genus Trichosporon—Tychosporon brassicae Trichosporon brassicae CGMCC No.0574 and CGMCC No.0573 are the only species in the genus Citeromyces; Trichosporon brassicae CGMCC No.0574 and Trichosporon laibacchii are different species in the same genus. These two bacterial strains have been deposited in the China Microorganism Culture Collection Management Committee on May 09, 2001, and the preservation numbers are respectively CGMCC No.0573 and CGMCC No.0574.

The above-mentioned strains have the following properties:

Morphological characteristics:

Saccharomyces solidis CGMCC No.0573: After being cultured in wort liquid medium at 30°C for 3 days, the bacterium presents spherical vegetative cells, does not produce candida, and contains one ascospore in each ascus. The nitrate reduction test is positive and does not produce candida. urease. The bacteria can ferment glucose, sucrose and maltose, but not lactose. The bacteria can assimilate succinic acid, lactic acid and ethanol, but cannot assimilate methanol, and weakly assimilate citric acid.

Trichospora brassicae CGMCC No.0574: After being cultured in glucose yeast extract peptone liquid medium at 25°C for 3 days, there are fungal filaments and arthrospores, and budding cells are rare; the cell size is (3-4.5)×(5-20 ) μm, with bacterial film formation and flocculent sediment. After culturing in glucose yeast extract peptone agar medium at 25°C for one month, the streaked culture was off-white, with zigzag lines, velvety, tough texture, and hyphae formed at the edge. When cultured on cornmeal agar Dalmau plates, a large number of mycoceles are produced. The bacteria cannot ferment sugars.

According to the literature: "The yeasts: a taxonomic study" (Kurtzman et al, 1998) and "Characteristics and Identification of Yeast" (Barnett et al, 1983: Yeasts-characteristics and identification, Qingdao Ocean University Press) provided the identification method And the above-mentioned test result, show that Saccharomyces CGMCC0573 belongs to Saccharomyces genus, and there is only one species of Citeromyces matriensis in this yeast group; The species was identified and named by the Institute of Microbiology, Chinese Academy of Sciences on May 9, 2001 ("2001 Weijianzi No. 119"). Through conventional fermentation methods, the above-mentioned Saccharomyces solidis CGMCC No.0573 and T. brassicae CGMCC No.0574 strains can be cultivated.

The above strains were obtained in Shanghai, and the screening method is as follows:

Take 1g of fresh soil sample and add it to 50ml screening medium (containing 0.2% (NH ₄ ) ₂ SO ₄ , 0.2% K ₂ HPO ₄ , 0.05% NaCl and 0.05% MgSO ₄ ·7H ₂ O), and add a small amount of ketone The Tween-80 emulsion or ethanol solution of ibuprofen ethyl ester is used as the only carbon source, and carries out two rounds of enrichment culture; Thin plate chromatography is carried out after enrichment, gets the nutrient solution that has ester hydrolyzate (ketoprofen), Dilute and smear the plate (the composition of the medium is the same as above) for culture, take a single colony with a transparent circle, and culture it with rich medium (for example: glucose 1%, peptone 0.5%, yeast extract 0.5%) in a shaking tube for 24 hours, then add ketone base The emulsion (10mM) of ibuprofen ethyl ester, after converting 24h, extracts product acid and residual ester with the ethyl acetate of equal volume, makes HPLC analysis (uses the ChiracelOJ of ChiracelOJ of Japan Daicel Company, φ 0.46cm ×25cm, the mobile phase is n-hexane/isopropanol/acetic acid=90:10:0.1), and the conversion rate of the substrate and the enantiomeric excess value (ee _p ) of the product were calculated. Finally, two microorganisms that selectively hydrolyze (R)- and (S)-ketoprofen ethyl ester were selected from more than 500 candidate strains: Citeromyces matriensis CGMCCNo.0573 and Trichospora brassicae Trichosporon brassicae CGMCC No. 0574.

The method for preparing optically pure 2-aryl propionic acid using the above-mentioned microorganisms comprises the steps in turn:

(1) Adopt conventional method to carry out bacterial classification and cell culture:

Preparation of bacterial strains: respectively put Citeromyces matriensis CGMCC No.0573 and Trichosporon brassicae CGMCC No.0574 in sterilized (121°C, 20-40min) rich medium (for example: glucose 1% , peptone 0.5%, yeast extract 0.5%, agar 1.5%) streaked on the plate, cultured at 25-30°C for about 2 days, picked a single colony, and used it as a seed for slant culture (cultivation conditions are the same as above), and stored it after about 2 days Store in a refrigerator at 4°C for later use.

Cell culture: Pick the seeds cultivated on the above slant, inoculate them into a 100mL small shaker flask filled with 20mL liquid medium (the composition is the same as above, but without adding agar), and inoculate them at an appropriate temperature (20-50°C, preferably 25-40°C). °C) and rotating speed (for example, 60-300r/min, generally 120-180r/min) on a shaker for 12-18h, inoculate in an appropriate proportion (such as 5%) in a 500mL large shaker flask containing 100mL growth medium, After culturing on a shaker (30° C., 150 r/min) for 24-30 hours, the supernatant was removed by centrifugation at 4° C. and 8000 r/min, and the obtained cells were washed once with physiological saline (0.85% NaCl).

(2) Transformation of the substrate: suspend the cultured cells of the strain with the preservation number CGMCC No.0574 (containing S-specific esterase in the cell) in phosphoric acid at a concentration of 10g to 200g (wet weight)/L In the potassium buffer solution, the concentration of the potassium phosphate buffer solution is 20-100mmol/L, the pH is 6-11, preferably pH=8-9, and the emulsification solution containing the substrate ester of the emulsifier is added, and the preferred emulsifier is One of Tween-80, polyvinyl alcohol or nonylphenol polyoxyethylene ether and the mixture thereof, added in an amount of 1-20g/L, shaken and reacted on a shaking table at 20-50°C for 1-5 days, The reaction solution was acidified with hydrochloric acid to a pH of about 2-3, then extracted with an equal volume of ethyl acetate, allowed to stand, and the phases were separated to collect the organic phase containing acid and ester. Sampling from the organic phase is performed by chiral high-performance liquid chromatography, and the conversion rate of the substrate and the enantiomeric content (ee _p ) of the product are calculated;

The substrate ester is one of the esters formed by 2-aryl propionic acid and C ₁ -C ₈ fatty alcohol.

(3) Separation of products: adding dilute lye to the above-mentioned organic extract containing acid and ester for back extraction, the aqueous phase containing (S)-acid and the organic phase containing (R)-ester can be obtained.

The dilute lye includes NaOH or/and NaHCO ₃ , the concentration of which is 0.01-1.0 mol/L, and the amount added so that the final pH is 10-12 is more appropriate;

Add hydrochloric acid to the aqueous phase to acidify to a pH of 2-3, let stand overnight and then filter to obtain an optically pure (S)-type crystal product of a single enantiomer of 2-arylpropionic acid.

The organic phase containing (R)-ester is heated and evaporated, and the solvent is removed to obtain (R)-ester.

(4) Secondary resolution: the (R)-ester obtained by the above extraction and separation is added with an emulsifier such as Tween-80, polyvinyl alcohol or nonylphenol polyoxyethylene ether and a mixture thereof, and the emulsifier The addition amount is 1-20g/L to make (R)-ester emulsion. The cells (containing R-specific esterase) cultured by the strain with the preservation number CGMCC No.0573 were used as catalysts, suspended in potassium phosphate buffer at a concentration of 10g-200g (wet weight)/L, and potassium phosphate buffer The concentration of the solution is 20-100mmol/L, and the pH is 6-11, preferably pH=8-9. Mix the (R)-ester emulsion with the cell suspension in equal volumes, shake and react on a shaking table at 20-50°C for 1-5 days, then acidify with hydrochloric acid to a pH of about 2-3, and then add an equal volume of acetic acid Extract with ethyl ester, stand still, separate the phases, and collect the organic phase containing acid and ester. Sampling from the organic phase is analyzed by chiral high-performance liquid chromatography, and the conversion rate of the substrate and the enantiomeric excess value (ee _p ) of the product are calculated;

Add dilute lye (control the final pH at 10-12) to the organic extract containing acid and ester for back extraction to obtain an aqueous phase containing (R)-acid and an organic phase containing (S)-ester. Add hydrochloric acid to the separated aqueous phase to pH = 2-3, then stand overnight and filter to obtain optically pure (R)-arylpropionic acid crystals.

According to the present invention, the racemic 2-aryl propionate can also be hydrolyzed to a conversion rate of 30-40% with cells of R-specific esterase-containing Saccharomyces solidis CGMCC No.0573, and after extraction Separation of (R)-type 2-aryl propionic acid with higher optical purity and (S)-2-aryl propionate with lower optical purity; The remaining (S)-enantiomer-enriched ester of the cell-catalyzed hydrolysis reaction of Trichosporium yeast CGMCC No.0574 was then subjected to alkaline extraction and acidification crystallization to obtain optically pure (S)-type 2-aryl propionic acid crystals.

According to the present invention, preferably, the cultured cells are shaken in a buffer solution containing acetone or isopropanol for 1-15 hours, and then used for the above-mentioned catalytic reaction.

By adopting the biotransformation process of the present invention, not only (S) and (R)-type 2-aryl propionic acid single enantiomer products with high optical purity can be obtained simultaneously, which meets the needs of the pharmaceutical or pesticide industry for different purposes , and the ethyl 2-aryl propionate as the reaction substrate is easy to synthesize without using expensive or toxic reagents, thereby reducing the cost of raw materials; in addition, it also saves the racemization and recycling of the remaining esters of the reaction, thereby The operation steps are simplified and the utilization rate of raw materials is improved.

Detailed ways

The specific implementation manner of the present invention will be described in detail below through examples.

Example 1-6

Put 3L growth medium (1% glucose, 0.5% peptone, 0.5% yeast extract) in a 5L fermenter, insert CGMCC No.0574 seed culture solution according to 5% inoculum after sterilization, and keep the temperature at 30°C 1. Under the condition that the stirring speed is 600r/min and the ventilation rate is 0.4vvm, after about 12 hours, the glucose is basically exhausted, the cell density reaches 8.5g (dry weight)/L, and the esterase activity reaches 10.3U/L. At this time, add glucose to 5g/L, the bacteria continue to grow and produce enzymes, and the bacterial concentration can be increased by 60% and the enzyme activity can be increased by about 50% when put in the tank for 24 hours.

Take 100 mL of the above-mentioned culture solution, collect the bacteria by centrifugation, wash with physiological saline, suspend in 20 mL of phosphate buffer (50 mM, pH=7.0), add 0.1 g of Tween-80, and then add ketoprofen with different The ester (20mM) obtained by alcohol synthesis was incubated and reacted on a shaking table at 30°C and 120r/min, and the results are shown in the table below.

Table 1 The hydrolysis results of different esters of ketoprofen by Trichospora brassicae CGMCC No.0574.

Ketoprofen relative rate conversion rate product configuration enantioselectivity

Various esters (%) (%) and their content (%) (E value) Example 1 Methyl ester 211 49 R (93) 34 Example 2 Ethyl ester 100 45 5 4 Ethyl chloride S (96) 67 49 s (80) 7.0 Example 4 isopen esters 38 44 R (85) 9.7 Example 5 orthodoxyl ester 47 37 s (70) 2.9 Example 6 26 23 s (65) 2.0

Example 7

Inoculate CGMCC No.0573 into a shaking tube containing 4 mL of growth medium (glucose 1%, peptone 0.5%, yeast extract 0.5%), culture at 30°C and 120 r/min for 42 hours, then add 20 μl of 2M The ethanol solution of ketoprofen ethyl ester, after converting for 48h, after extracting with 4mL ethyl acetate, carried out HPLC analysis, the result obtained (R)-ketoprofen, enantiomeric purity is 96.8%, product The rate is 27%.

When the substrate ketoprofen ethyl ester was added in the conversion reaction system with Tween-80 emulsion, the reaction speed was obviously accelerated, and the conversion ratio reached 31% in the time of 16h, and the product (R)-ketoprofen The enantiomeric purity is 96.5%.

Example 8

Take 10 mL of Trichospora brassicae CGMCC No.0574 culture solution after culturing for 24 h in a test tube, centrifuge and wash to collect the obtained bacteria, resuspend in 2 mL of phosphate buffer containing 1% acetone or isopropanol (50 mM, pH 7 .0), shake on a shaker for 12 hours, centrifuge, wash away the treatment reagent, then incubate in 2 mL of phosphate buffer with 10 mM substrate emulsion (containing 0.5% Tween-80), and measure the hydrolysis of the ester relative rates and enantioselectivities. Results Compared with the fresh cells without permeabilization treatment, the initial speed of the cells treated with acetone increased by 61%, while the reaction speed of the cells treated with isopropanol increased to 2.35 times. There was no significant change in the enantioselectivity, and the enantiomeric purity of the product remained at 96-97%.

When the above-mentioned permeabilization buffer was replaced by pH=7.0 phosphate to pH=10 glycine-NaOH buffer, the reaction rate was increased by 75%. The pH=10 buffer was treated with 1% isopropanol for different periods of time, and the best result was 10 hours. Under these conditions, the treatment effect of 2% isopropanol was better than that of 1%, and the reaction speed was more than three times that of the control group. When the concentration of isopropanol is fixed at 2% and the concentration of cells is changed, the effect of treatment is also significantly affected, and the cell amount of 30g (wet weight)/L is the best.

Example 9

Cultivate the cells of CGMCC No.0573 and CGMCC No.0574 in a 500mL large shake flask (conditions as above), centrifuge and wash for later use.

Take 4g of Trichospora brassicae CGMCC No.0574 cells and treat them with (30g/L cells, 2% isopropanol, pH=10) for 10h, then suspend them in 50mL of phosphate buffer (100mM, pH8.0) , add 1.4g ketoprofen ethyl ester (100mM) and 0.25g Tween-80 (0.5%), after 36h at 30°C and 120r/min insulation reaction, add a few drops of concentrated hydrochloric acid to acidify to pH=2, then The generated ketoprofen acid and the remaining ester were extracted with 50 mL ethyl acetate, and 1 mL was sampled for HPLC analysis. As a result, the yield of (S)-acid was 30%, and the enantiomeric purity was 95.5%. The rest of the extract was back-extracted with 0.1N NaOH solution to obtain the salt solution of (S)-ketoprofen, which was acidified with hydrochloric acid to obtain the precipitate of (S)-ketoprofen, and then further crystallized (NaHCO ₃ /HAc) After that, the optical purity increased to over 98%.

The organic phase obtained after the above stripping was evaporated and concentrated to obtain (R)-ketoprofen ethyl ester (enantiomeric content: 78%). Suspend 0.46 g of the ester in 50 mL of phosphate buffer (100 mM, pH=7.0), add 0.25 g of Tween-80 and 4 g of wet cells of Saccharomyces solidis CGMCC No.0574, and incubate at 30 °C and 120 r/min for 90 h , It is measured that the yield of (R)-ketoprofen is 40%, and the enantiomer purity is 97.8%; after separation and crystallization, the content of (R)-enantiomer in the product exceeds 99%.

According to the technical scheme and the examples disclosed by the present invention, relevant engineers and technicians can easily use the two strains of yeast and their cultivation and transformation techniques of the present invention for other types of 2-aryl propionic acid (such as enantiomeric resolution of naproxen and ibuprofen, etc.) and 2-aryloxypropionic acids.

Claims (10)

1. Citeromycesbaodingensis bacterial strain CGMCC No.0573.

2. Trichosporon brassicae bacterial strain CGMCC No.0574.

3. a method for preparing optical purity 2-arylpropionic acid is characterized in that, this method comprises the steps:

(1) adopt conventional method that the bacterial strain of claim 1 and claim 2 is cultivated;

(2) with preserving number be the bacterial strain institute cultured cells of CGMCC No.0574, at pH is in the damping fluid of 6-11, and with the emulsion oscillatory reaction of the substrate ester that contains emulsifying agent, then reaction solution being acidified to pH with acid is 2～3, use ethyl acetate extraction, collect the organic phase that contains acid and ester;

Said substrate ester is 2-arylpropionic acid and C ₁～C ₈Fatty Alcohol(C12-C14 and C12-C18) forms a kind of in the ester;

(3) separation of product: add the alkali lye back extraction in the acid and the organic phase of ester in above-mentioned containing, the organic phase that obtains to contain the water of (S)-acid and contain (R)-ester, the alkali lye add-on is so that final pH is 10～12;

Is 2-3 with aqueous phase as acidified to pH with acid, the single enantiomer 2-arylpropionic acid of collection optically pure (S)-type wherein;

The organic phase heating evaporation that will contain (R)-ester is removed solvent, obtains (R)-ester.

(4) secondary splits: with the preserving number be CGMCC No.0573 bacterial strain institute cultured cells pH be in the damping fluid of 6-11 with the emulsion oscillatory reaction that contains (the R)-ester of emulsifying agent, being acidified to pH with acid is 2～3, use ethyl acetate extraction, collect the organic phase that contains acid and ester;

Add alkali lye in organic phase, the control final pH is collected water that contains (R)-acid and the organic phase that contains (S)-ester at 10-12, to pH=2-3, collects optical purity (R)-2-arylpropionic acid wherein with the acidifying water.

4. method as claimed in claim 3 is characterized in that, said damping fluid is a potassium phosphate buffer, and concentration is 20～100mmol/L, and the cell in the damping fluid is counted 10g～200g/L with weight in wet base, and pH of buffer is 8-9.

5. method as claimed in claim 3 is characterized in that, said emulsifying agent is a kind of and composition thereof in tween-80, polyvinyl alcohol or the polyoxyethylene nonylphenol ether, and add-on is 1～20g/L.

6. method as claimed in claim 3 is characterized in that, said alkali comprises NaOH or/and NaHCO ₃, concentration of lye is 0.01～1.0mol/L.

7. method as claimed in claim 3 is characterized in that, said acid is hydrochloric acid.

8. method as claimed in claim 3 is characterized in that, the oscillatory reaction temperature is 20～50 ℃, and the time is 1～5 day.

9. a method for preparing optical purity 2-arylpropionic acid is characterized in that, this method comprises the steps:

(1) adopt conventional method that the bacterial strain of claim 1 and claim 2 is cultivated;

(2) racemic 2-arylprop acid esters is hydrolyzed into the 30-40% transformation efficiency with the cell catalysis of Citeromycesbaodingensis CGMCC No.0573 earlier, go out higher (R)-type 2-arylpropionic acid of optical purity and lower (the S)-2-arylprop acid esters of optical purity through extracting and separating, and then with the ester of remaining (the S)-enantiomorph of the cell catalysis hydrolysis reaction part enrichment of Trichosporon brassicae CGMCC No.0574, through alkali lye extraction and acidizing crystal, obtain optically pure (S)-type 2-arylpropionic acid again.

10. as the arbitrary described method of claim 3～9, it is characterized in that, will cultivate the cell obtained oscillation treatment 1～15 hour in the buffered soln that contains acetone or Virahol.